Maintenance of a steady internal pH is a critical feature of cell physi- ology in many systems, including bacteria. In Escherichia coli, internal pH remains nearly constant over a wide range of external pH. Proton transport is known to be modulated by the electron transport chain, by the ATPase proton pump, and by cation exchange; metabolic consumption of acids also affects pH. It is not known, however, which features are essential to maintain internal pH at a defined level. In this project, E. coli mutants will be isolated that exhibit defective pH homeostasis. Selection procedures for internal -pH mutants will be based on the transmembrane Delta pH-dependent uptake of a membrane-permeable weak acid, and on the "proton suicide" treatment with bromide/bromate medium. Additionally, gene loci whose expression exhibits internal pH-dependence will be isolated as lacZ gene fusions inserted by Lambda placMu. Mutants obtained by both methods will be characterized with respect to: (a) pH homeostasis and membrane potential, using filtration assays; (b) behavioral responses to pH perturbation, and methylation levels of the pH-sensitive chemotaxis protein tsr; (c) genetic mapping and complementation with loci of known function in proton transport. Mutants with altered levels of internal pH, or with gene fusions whose expression is regulated by pH, should provide insight into the mechanism of pH homeostasis.